Use of taurolidine for treatment of leukemias

ABSTRACT

The invention relates to selective induction of cell death by apoptosis and applicability to treatment of leukemias.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of prior filed copending U.S.Provisional Application No. 60/047,642 filed May 22, 1997 and is a 371of PCT/U.S. Ser. No. 98/10494, filed May 22, 1998.

BACKGROUND OF THE INVENTION

Cell death proceeds by one of two mechanisms: necrosis or apoptosis. Innecrosis, the cells lyse and cytosolic components are released. Thereleased cytosolic components elicit severe inflammatory responses.Apoptosis does not result in the release of cytosolic contents, as thecell membrane remains intact even though its surface properties maychange. Apoptosis may include the break up of cells into apoptoticbodies, spherical pieces of cells in which the membrane still preventsthe release of cytosolic contents. Apoptotic cells and apoptotic bodiesare removed in the body by phagocytic cells which are believed torecognize the need to remove such cells by the changes in the outerleaflet of the membrane, in which phosphatidylserine is exposed.Apoptosis typically does not provoke inflammatory responses the waynecrosis does because in the former case, the cells are removed byphagocytosis before the cytosolic content is released.

Although cells undergoing apoptosis in vitro initially have intact cellmembranes, cells in advanced stages of apoptosis can exhibit loss ofmembrane integrity. This process is sometimes called “secondarynecrosis.” It can be observed owing to the absence of phagocytic cells,which in vivo would have removed the apoptotic cells and cell fragmentsbefore they could become necrotic.

When neoplastic (tumor) cells are present in the body, it is desirableto cause the death of such cells without causing the death of the normalcells which the patient needs to sustain his life. It is desirable tocause the death of neoplastic cells by inducing apoptosis, so that thecytosolic contents of the neoplastic cells are not released.

Antineoplastic drugs have been reported which kill tumor cells byinducing apoptosis. While some of these drugs have been successful intreating some types of cancer, the drugs have also been known to inducesevere side effects, such as cytotoxicity to normal cells byinterference with basic cellular functions such as protein synthesis orDNA replication. A few inducers of apoptosis in monocytes have beenreported. For example, human blood monocytes can undergo apoptosis whencultured in the absence of serum or stimulatory factors (which isimpossible to achieve in vivo). Mangan, et al., “Lipopolysaccharide,tumor necrosis factor-α, and IL-1β prevent programmed cell death(apoptosis) in human peripheral blood monocytes,” J Immunol 146:1541(1991). This process takes two to three days for approximately 50% ofthe monocytes to become apoptotic (Mangan, et al., “IL-4 enhancesprogrammed cell death (apoptosis) in stimulated human monocytes,” JImmunol 148:1812 (1992)), and apoptosis can be postponed bylipopolysaccharide (LPS), interleukin (IL)-1, and α-tumor necrosisfactor (TNFα). In addition, the anti-inflammatory cytokine IL-4 canenhance apoptosis in LPS-stimulated monocytes. Mangan, D. F. and Wahl,S. M., “Differential regulation of human monocyte programmed cell death(apoptosis) by chemotactic factors and pro-inflammatory cytokines,” JImmunol 147:3408-3412(1991). Apoptosis has also been induced in severaldifferent cell types by the use of a number of cytokines. However, thepotential use of cytokines for treatment of cancer in vivo has sufferedfrom drawbacks, since they have been reported to elicit a variety ofdeleterious effects, including shock, circulatory collapse and death. Inaddition, the manufacture of cytokines has been to date, complicated andexpensive since recombinant technology for manufacturing proteins is notan inexpensive proposition on a large scale basis.

Further adding to the complicated nature of leukemia treatment is thefact that there are many different types of leukemia. In viewing thescheme of hemopoiesis, pluripotent stem cells divide to form eitherlymphoid stem cells or myeloid stem cells. Lymphocytes are produced fromlymphoid stem cells, while monocytes and granulocytes such asneutrophils, eosinophils and basophils are produced from myeloid stemcells. Myeloid stem cells also give rise to erythrocytes andmegakaryocytes. Various leukemias resulting from these differentiatedcells include lymphocytic leukemia, monocytic leukemia, and myeloidleukemia. Treatment methodologies and prognosis differ depending on thespecific type of leukemia.

Particularly difficult to treat are myeloid and monocytic leukemias.Current treatment methods have achieved palliation and not cure. Forexample, patients having monocytic leukemia are generally thought tohave a low cure rate of less than ten percent. A true remission isimpossible to achieve because the Ph-positive clone persists in the bonemarrow, and intense chemotherapy treatments designed to eliminate orreduce the clone have only provided modest improvements in the length ofsurvival of these patients. Current chemotherapy is designed to keep thepatient asymptomatic for long periods of time by maintaining a totalwhite blood count within an acceptable range.

It is, therefore, desirable to find a new agent that could selectivelycause apoptosis of monocytic, myeloid, and leukemia cells withoutcausing severe side effects that accompany the administration oftraditional chemotherapeutic agents. It has now been found that a knowncomposition, taurolidine, can be used for the induction of apoptosis inmonocytic and myeloid cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting the expression of tissue factor (TF) bylipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells(PBMC) and Mono Mac 6 (MM6) cell line. PBMC at 1.5×10⁷ cells/ml (opensquares), or 5.6×10⁵ cells/ml (closed squares) were cultured in AIM-Vsupplemented with 0.01% bovine calf serum (AIM-V+0.01% BCS). Aftertreatment with 10 ng/ml LPS, samples were collected for ELISA at theindicated times. Data (mean±SD from samples assayed in quadruplicate)are from a representative experiment. In this experiment, uninduced MM6cells expressed 160 pg TF per 1×10⁶ cells, while induced PBMC expressedless than 1 pg TF per 1×10⁶ cells.

FIG. 2A and FIG. 2B are graphs depicting taurolidine inhibition ofLPS-induced TF expression by monocytic cells as determined by assayprocedures. FIG. 2A represents MM6 cells; FIG. 2B represents PBMC. Thecells were induced with LPS (hatched bars: 100 ng/ml LPS in RPMIsupplemented with 10% BCS (RPMI+10% BCS); solid bars: 10 ng/ml inAIM-V+0.01% BCS) in the presence of varying concentrations oftaurolidine, or with a dilution of the vehicle equivalent to that givento cells receiving 100 μg/ml taurolidine. After four hours, cells werecollected and lysates were prepared for TF ELISA. Data are expressed aspercent of TF antigen measured in cells treated with LPS alone (notaurolidine or vehicle). Experiments were performed with duplicatesamples and were repeated at least three times. Values shown aremean±SEM of all experiments.

FIG. 3A and FIG. 3B are graphs depicting taurolidine inhibition ofLPS-induced alpha tissue necrosis factor (TNFα) expression by monocyticcells as determined by assay procedures. FIG. 3A represents MM6 cells;FIG. 3B represents PBMC. The cells were induced with LPS (hatched bars:100 ng/ml LPS in RPMI+10% BCS; solid bars: 10 ng/ml in AIM-V+0.01% BCS)in the presence of varying concentrations of taurolidine. After fourhours, cell supernatants were collected for TNFα ELISA. Data areexpressed as percent of TNFα levels measured in cell cultures treatedwith LPS alone (no taurolidine or vehicle). Experiments were performedwith duplicate samples and were repeated at least three times. Valuesshown in FIG. 3A are from a representative experiment. Values shown inFIG. 3B are mean±SEM of three experiments.

FIG. 4A and FIG. 4B are graphs depicting cell viability and growth ratesfollowing taurolidine treatment as determined by assay procedures. MM6cells were treated with taurolidine as described in FIG. 3. Afteraliquots were collected for the lactate dehydrogenase (LDH) assay, theremainder of the culture was rinsed in Hank's balanced salt solution(HBSS) and resuspended in RPMI+10% BCS without taurolidine. Counts oftrypan blue-negative cells represented in FIG. 4A and total cell numberrepresented in FIG. 4B were recorded daily thereafter (with themeasurement at Day 1 being made immediately after the four hourtaurolidine treatment). Conditions were no treatment (open squares); 50μg/ml taurolidine (closed squares); 25 μg/ml taurolidine (open circles);and vehicle alone (closed circles). Cells were subcultured when theyreached a concentration of 6×10⁵ cells/ml. Data are mean±SEM of threeexperiments.

FIGS. 5A and 5B depict cell viability and growth rates for MM6 cell linefollowing taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 5A), and total cell number(FIG. 5B), were recorded daily thereafter (with the measurement at Day 1being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 5B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIGS. 6A and 6B depict cell viability and growth rates for K562 cellline following taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 6A), and total cell number(FIG. 6B), were recorded daily thereafter (with the measurement at Day 1being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 6B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIGS. 7A and 7B depict cell viability and growth rates for HL-60 cellline following taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 7A), and total cell number(FIG. 7B), were recorded daily thereafter (with the measurement at Day 1being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 7B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIGS. 8A and 8B depict cell viability and growth rates for REH cell linefollowing taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 8A), and total cell number(FIG. 8B), were recorded daily thereafter (with the measurement at Day 1being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 8B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIGS. 9A and 9B depict cell viability and growth rates for Jurkat cellline following taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 9A), and total cell number(FIG. 9B), were recorded daily thereafter (with the measurement at Day 1being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 9B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIGS. 10A and 10B depict cell viability and growth rates for ECV304 cellline following taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 10A), and total cell number(FIG. 10B), were recorded daily thereafter (with the measurement at Day1 being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 10B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIGS. 11A and 11B depict cell viability and growth rates for GM5387 cellline following taurolidine treatment, in terms of % trypan blue negativecells and % cell number, respectively. Cells were treated with variousconcentrations of taurolidine for 4 hours in AIM-V+0.01% BCS. Afteraliquots were collected for LDH assay, the remainder of the culture wasrinsed in HBSS and resuspended in RPMI+10% BCS without taurolidine.Counts of trypan blue-negative cells (FIG. 11A), and total cell number(FIG. 11B), were recorded daily thereafter (with the measurement at Day1 being made immediately after the 4 hour taurolidine treatment).Conditions were: no treatment (open circles); 25 μg/ml taurolidine(closed circles); 50 μg/ml taurolidine (open squares); 100 μg/mltaurolidine (closed squares); and vehicle alone (closed triangles).Doubling times were determined under each condition, and expressed as apercentage of the rate of doubling of untreated cells. These values arelocated to the right of the growth curves in (FIG. 11B). Cells weresubcultured when they reached a concentration of 6×10⁵ cells/ml. Dataare mean±SEM of three experiments.

FIG. 12A, FIG. 12B, FIG. 12C, and FIG. 12D are flow cytometric analysesdepicting changes in cell morphology following taurolidine treatment.MM6 and PBMC were treated for three hours with taurolidine, and thentreated and untreated control cells were processed for flow cytometry.Monocytes in PBMC samples were identified by staining withFITC-conjugated anti-CD14, and lymphocytes by FITC-conjugated anti-CD3and PE-conjugated anti-CD19. FIG. 12A represents untreated MM6 cells;FIG. 12B represents MM6 cells treated with 50 μg/ml taurolidine; FIG.12C represents untreated PBMC; and FIG. 12D represents PBMC treated with75 μg/ml taurolidine. M=monocytes; L=lymphocytes.

FIG. 13 is an agarose gel depicting DNA fragmentation in response totaurolidine treatment as determined by assay procedures. MM6 cells wereincubated with taurolidine or vehicle in RPMI+10% BCS for four hours.Cells were harvested and low molecular weight DNA extracted as describedbelow. C=control cells without taurolidine or vehicle treatment;T=treated with 50 μg/ml taurolidine; V=treated with vehicle alone; andMW=molecular weight markers.

FIG. 14A and FIG. 14B are terminal deoxytransferase-mediateddeoxyuridine-triphosphate-biotin nick end labeling assay (TUNEL)analyses of control cells and taurolidine-treated PBMC, respectively.Cells were incubated with 100 μg/ml taurolidine for six hours andprocessed for TUNEL analysis as given below. Parallel samples werestained with FITC-conjugated anti-CD14 and propidium iodide (PI) asdescribed below. Ten thousand events were recorded per sample. TUNELsignal (x-axis) is a measure of DNA fragmentation, which FSC (y-axis) isrelated to cell size. TUNEL-negative lymphocytes are bounded by therectangle R1, while TUNEL-negative monocytes (CD14 positive cells) arebounded by the rectangle R2. The vertical line indicates demarcationbetween TUNEL-negative and TUNEL-positive cells. There were minimalnumbers of PI-positive cells in the six hour samples (see Table IV).

FIGS. 15A, 15B, and 15C depict TUNEL analysis of PBMC treated withtaurolidine in whole blood, for control cells, taurolidine-treatedcells, and anisomycin-treated cells, respectively . Cells were incubatedwith 500 μg/ml taurolidine for 4 hours, resuspended in fresh medium,incubated for two hours further and processed for TUNEL analysis.Parallel samples were stained with FITC-conjugated anti-CD14 and PI asdescribed herein. Ten thousand events were recorded per sample. TUNELsignal (x -axis) is a measure of DNA fragmentation, while FSC (y-axis)is related to cell size. TUNEL-negative lymphocytes are bounded byrectangle R 1, while TUNEL-negative monocytes (CD 14 positive cells) arebounded by rectangle R2. R3 bounds the TUNEL-positive population ofcells. There were minimal numbers of PI-positive cells in 6 hour samples(see Table VI).

DETAILED DESCRIPTION

Taurolidine (taurolin; tauroline;4,4′-methylenebis(perhydro-1,2,4-thiadiazin 1,1-dioxide) is disclosedfor the first time as an agent for treating patients inflicted withmonocytic and/or myeloid leukemias. Taurolidine is administered byinjection in solution (intravenously or intraperitoneally) to afflictedpatients in an amount effective to cause apoptosis of monocytic and/ormyeloid cells. The cells involved in the monocytic or myeloid leukemiadisease are thus attacked and die via apoptosis.

Taurolidine has been reported as a useful agent for other applications,and thus, in vitro and clinical data is available on its physiologicaleffects, even though there are no previous reports on its use as anapoptosis-inducer in monocytes, granulocytes, or leukemia cells. Notoxic effects on epithelial cells were reported after two hour treatmentwith 5 mg/ml taurolidine, (Gorman, et al., “Reduced adherence ofmicroorganisms to human mucosal epithelial cells following treatmentwith taurolin, a novel antimicrobial agent,” J Appl Bacteriol 62:315(1987)), and previous studies on monocytes reported that taurolidine wasapparently non-toxic since there was no increase in LDH release by cellsafter 24-hour taurolidine treatment. Bedrosian, et al., “Taurolidine, ananalogue of the amino acid taurine, suppresses interleukin 1 and tumornecrosis factor synthesis in human peripheral blood mononuclear cells,”Cytokine 3:568 (1991); Dofferhoff, et al, “The release of endotoxin fromantibiotic-treated Escherichia coli and the production of tumor necrosisfactor by human monocytes,” J Antimicrob Chemother 31:373 (1991).

In previous reports on other applications of taurolidine, such as for anagent to address septicemia, plasma concentrations of taurolidine (orits initial metabolite) achieved in clinical use range from 20 to 100μg/ml in septic patients or normal volunteers. Browne, M. K.,“Pharmacological and clinical studies with taurolin.” In Taurolin, EinNeues Konzept zur Antimikrobiellen Chemotherapie ChirurgischerInfektionen, Brückner, W. L. and Pfirrmann, R. W. (eds),München-Wien-Baltimore, Urban & Schwarzenberg, p. 3, 1985; Nitsche, etal., “Investigations of endotoxin inactivation in plasma. Preliminaryresults of a controlled randomized study on taurolidine as asupplementary therapeutic agent in septicemia.” In Emergency SurgeryTrends, Techniques, Results. Proceedings of the 7th InternationalCongress of Emergency Surgery, Schweiberer, L. and Eitel, F. (eds),Munich, Zuckschwerdt, p.185, 1985). Taurolidine treatment in humans hasnot been associated with toxicity, even when septic patients havereceived up to 20 grams for 2-5 consecutive days. Willatts, et al.,“Effect of the antiendotoxic agent, taurolidine, in the treatment ofsepsis syndrome: a placebo-controlled double-blind trial,” Crit Care Med23:1033 (1995); Johnston, et al., “Taurolin for the prevention ofparenteral nutrition related infection: antimicrobial activity andlong-term use,” Clin Nutrition 12:365 (1993).

It has been found that concentrations of taurolidine in the range of 50to 100 μg/ml induced apoptosis in 75% of peripheral blood monocytesincubated in culture medium within six hours. Further, 100 μg/mltaurolidine induced apoptosis in 92% of granulocytes incubated inculture medium within six hours. Preferred therapeutic dosages fortreatment of leukemias are from about 10 to about 500 mg/kg body weight.Most preferred is a dose that results in a 200 μg/ml concentration inthe plasma, which is typically about 150 mg/kg body weight.

Taurolidine can be administered intravenously or intraperitoneally. Fortreatment of the various leukemias, an effective amount of taurolidineto cause apoptosis of the affected cells is used and can be monitored byperiodic tests on samples of the patient's blood where cells can beobserved for evidence of apoptosis using any technique such as TUNELanalysis, DNA fragmentation, or with other suitable markers ofapoptosis.

In our studies, taurolidine was used in the form of a 2% w/v solution ina vehicle containing 2% dextrose and 5% polyvinylpyrrolidone. A solutionconsisting of the vehicle alone was obtained from Wallace Laboratories(Cranbury, N.J.) for the preparation of a vehicle control. Reagents andsupplies were obtained as follows: Histopaque, propidium iodide (PI),Lactate Dehydrogenase (LDH) Kit, rabbit brain thromboplastin,anisomycin, and dimethylsulfoxide (DMSO) from Sigma Chemical Co. (St.Louis, Mo.); 6% dextran 70 in 0.9% NaCl (6% dextran) from McGaw, Inc.(Irvine, Calif.); RPMI, Eagle's minimum essential medium (MEM), andHank's balanced salt solution (HBSS) from Mediatech, Inc. (Herdon, Va.);medium M199 from Bio Whittaker, Inc., (Walkersville, Md.); bovine calfserum (BCS) from HyClone Laboratories, Inc. (Logan, Utah.); 25% humanserum albumin (HSA) from the American Red Cross Blood Services(Washington, D.C.); AIM-V medium from Gibco BRL (Grand Island, N.Y.);LPS (E. coli 0111:B4) and bovine serum albumin (fatty acid free) fromCalbiochem Corp. (La Jolla, Calif.); FITC-conjugated anti-CD14antibodies, FITC-conjugated anti-CD3/PE-conjugated anti-CD19 Simultest,and PE-conjugated anti-CD16 from Becton Dickinson (Parsippany, N.J.);and the TUNEL assay system (In Situ Cell Death Detection Kit) fromBoehringer Mannheim (Indianapolis, Ind.).

Numerous cell lines have been examined and are listed in Table I. MM6cells, derived from the peripheral blood of a patient with acutemonoblastic leukemia (M5), were a generous gift of Dr. H. ZieglerHeitbrock. The K562 cell line, developed from cells from pleural fluidof a patient with chronic myeloid leukemia in blast crisis, was obtainedfrom the American Type Culture Collection (ATCC) (Rockville, Md.). K562cells can be induced to differentiate into precursors of the monocytic,granulocytic, and erythrocytic series. HL-60 cells, established fromperipheral blood leukocytes of a patient with acute promyelocyticleukemia, were obtained from ATCC. HL-60 cells display surface receptorsand react with cytochemical stains specific for granulocytic cells. REHcells, originated from acute lymphoblastic leukemia cells, were obtainedfrom ATCC. REH cells have a non-T, non-B phenotype. Jurkat is a humanleukemic T cell line available from ATCC. ECV304, a spontaneouslytransformed immortal endothelial cell line derived from a humanumbilical cord, was obtained from

TABLE I Cell Lines Tested Human Cell Line Derived from a Patient withHL-60 acute myeloid leukemia K-562 chronic myelogenous leukemia Mono Mac6 monocytic leukemia REH acute lymphocytic leukemia Jurkat humanleukemic T cell line ECV304 human endothelial cell line (spontaneouslytransformed) GM5387 human fetal fibroblast cell line

ATCC. The human fibroblast cell line GM5387, derived from tissueobtained by fetal lung biopsy, was obtained from NIGMS Human GeneticMutant Cell Repository.

All cells were cultured in a 5% CO₂ atmosphere at 37° C. MM6 cells weregrown in RPMI supplemented with 10% BCS, 2 mM L-glutamine, and 50 μg/mlgentamicin (RPMI+10% BCS). In some experiments, MM6 cells were rinsedonce with HBSS without Ca⁺⁺ or Mg⁺⁺ and once with AIM-V before finallybeing resuspended in AIM-V supplemented with 0.01% BCS (AIM-V+0.01%BCS). AIM-V has been shown to support long-term culture of macrophages(Helsinki, et al., “Long-term cultivation of functional humanmacrophages in teflon dishes with serum-free media,” J Leuk Biol 44:111(1988)), and in the present experiments, MM6 cells cultured in AIM-V forat least ten days maintained high viability and exhibited growth ratesonly slightly slower than cells grown in RPMI supplemented with 10% BCS.AIM-V was supplemented with a low amount of BCS (0.01%) in order toprovide a source of LPS-binding protein, which enhances the response ofmonocytic cells to LPS. Ulevitch, R. J. and Tobias, P. S., “Recognitionof endotoxin by cells leading to transmembrne signaling,” Curr OpinImmunol 6:125 (1994). HL-60, REH, and Jurkat were cultured in RPMIsupplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mML-glutamine, and 50 μg/ml gentamicin. ECV304 were cultured in M199supplemented with 10% FBS, mM L-glutamine, and 50 μg/ml gentamicin.GM5387 was cultured in MEM supplemented with 20% FBS, 2 mM L-glutamine,and 50 μg/ml gentamicin.

Human peripheral blood mononuclear cells (PBMC) were isolated fromheparinized blood by centrifugation using Histopaque as permanufacturer's instructions. The isolated PBMC were suspended in RPMIsupplemented with 10% BCS and were used in experiments immediatelythereafter. As determined by flow cytometry, PBMC were typically 11±2%monocytes and 80±13% lymphocytes, with the remaining cytometric eventsconsistent in size with platelets and cell aggregates.

Human granulocytes were isolated from heparinized blood by dilutingblood 1:1 with 6% dextran to sediment RBC, then centrifuging to obtainleukocyte rich plasma. The remaining RBC were lysed by 20 secondincubation with 0.2% NaCl. After lysis, NaCl was added to a finalconcentration of 0.9%, the sample was centrifuged, and the cell pelletresuspended in HBSS containing 0.5% HSA. An equal volume of Histopaque(Sigma) was layered underneath the cell suspension. Upon centrifugation,the PBMC remained above the Histopaque layer, while the granulocyteswere sedimented. Purity of collected granulocytes was assayed by flowcytometry using PE-conjugated anti-CD19.

Tissue Factor and TNFα Expression

The induction of tissue factor (TF) expression by bacterial LPS wasdemonstrated in numerous cell lines including human peripheral bloodmononuclear cells, human monocytic cell line Mono Mac 6 (MM6), andgranulocytes. The induction of tissue factor expression was blocked bytreatment with taurolidine.

For induction of TF or TNFα expression, MM6 cells or PBMC were culturedeither in RPMI supplemented with 10% BCS or in AIM-V+0.01% BCS, andstimulated by adding LPS to final concentrations of 10 to 100 ng/ml.Cells treated with taurolidine received final concentrations of 10 to100 μg/ml taurolidine, with parallel cultures receiving either notaurolidine or a comparable dilution of the vehicle alone. Except asnoted, taurolidine and LPS were added to the cell culturessimultaneously. For TF determination, cells and culture media togetherwere treated by adding Triton X-100 (1% final) and EDTA (7 mmol/Lfinal), and the resulting lysates assayed by ELISA. Rezaie, A. R., etal., “Expression and purification of a soluble tissue factor fusionprotein with an epitope for an unusual calcium-dependent antibody,”Protein Expression and Purification 3:453 (1992). For TNFαdetermination, cells were removed by centrifugation for 5 min at 250×g,and the supernatants were assayed by ELISA. Houston, et al.,“Endothelial cells and extracellular calmodulin inhibit monocyte tumornecrosis factor release and augment neutrophil elastase release,” J BiolChem 272:11778-11785 (1997). In order to examine if taurolidineinterfered with ELISA measurement of TF, taurolidine was added tolysates of cells that had previously not been treated with the drug.Taurolidine had no effect on the measurement of TF levels.

A number of previous studies have shown that peripheral blood monocytesrespond to LPS stimulation by expressing TF, with maximal expressiontypically occurring four to six hours after LPS treatment. Schwartz, etal, “Murine lymphoid procoagulant activity induced by bacteriallipopolysaccharide and immune complexes is a monocyte prothrombinase,” JExp Med 155:1464 (1982); Gregory, et al., “Regulation of tissue factorgene expression in the monocyte procoagulant response to endotoxin,” MolCell Biol 9:2752 (1989). Furthermore, monocytes are the only cell typepresent in PBMC preparations that are capable of expressing TF. Whencultured in AIM-V+0.01% BCS, MM6 cells and PBMC responded in a similarfashion to LPS stimulation (FIG. 1). For MM6 cells, maximal TFexpression was achieved with 10 ng/ml LPS, resulting in an 11-foldinduction of TF expression compared to unstimulated cells. For PBMC,maximal TF expression was >30-fold higher than in unstimulated cells.When MM6 cells were cultured in RPMI+10% BCS, similar time courses ofLPS-induced TF expression were observed, although maximal induction ofTF expression was achieved with 100 ng/ml LPS, resulting in a 4.3-foldinduction of TF expression compared to unstimulated cells.

The ability of taurolidine to inhibit LPS-induced TF expression in MM6and PBMC was examined. Taurolidine at concentrations of 50 μg/ml orhigher completely blocked the induction of TF expression in MM6 cells byLPS, while vehicle alone (used at a dilution comparable that of 100μg/ml taurolidine) had no effect (FIG. 2A). Lower concentrations oftaurolidine (10 or 25 μg/ml) had little or no effect on TF expression byMM6 cells. TF expression by LPS-stimulated PBMC was also inhibited bytaurolidine (FIG. 2B), although slightly higher concentrations wererequired (i.e., 75 μg/ml taurolidine was required for completeinhibition of TF expression, compared with 50 μg/ml for MM6 cells).Taurolidine exhibited similar effectiveness in blocking TF expressionwhen MM6 cells or PBMC where cultured in either RPMI+10% BCS or inAIM-V+0.01% BCS.

Some of the immunomodulatory effects of taurolidine have been proposedto result from its breakdown product, taurine. William, et al.,“Taurolidine, an antilipopolysaccharide agent, has immunoregulatoryproperties that are mediated by the amino acid taurine,” J Leuk Biol58:299(1995). Therefore, in order to examine this potential mechanism ofaction of taurolidine, we performed experiments as above using taurineat 44 μg/ml (equivalent in molar concentration to that which would begenerated from the complete breakdown of 50 μg/ml taurolidine) andhigher. Taurine had no effect on TF expression by MM6 cells, even atconcentrations up to 0.5 mg/ml. Another proposed mechanism of action oftaurolidine is via direct inactivation of LPS. Pfirrnann, R. W.,“Taurolin: ein neues konzept zur antimikrobiellen chemotherapiechirurgischer infecktionen einfürhrung und iübersicht.” In Taurolin, EinNeues Konzept zur Antimikrobiellen Chemotherapie ChirurgischerInfektionen, Brückner, W. L. and Pfirmann, R. W. (eds),München-Wien-Baltimore, Urban & Schwarzenberg, p. 3, 1985. Accordingly,whether or not pre-incubation of cells with taurolidine would affecttheir subsequent response to LPS stimulation was examined. Similarreductions in TF expression were observed when cells were pre-exposed to100 μg/ml taurolidine for thirty minutes, rinsed, and then exposed toLPS, compared to experiments in which taurolidine and LPS were addedconcurrently. Thus, direct contact between taurolidine and LPS was notnecessary for taurolidine to block the subsequent induction of TFexpression by LPS.

Taurolidine inhibited LPS-induced secretion of TNFα by MM6 cells andPBMC (FIG. 3). MM6 cells were slightly more sensitive to taurolidine,since maximal inhibition was obtained with 75 μg/ml taurolidine, while100 μg/ml taurolidine was required to maximally inhibit TNFα secretionby PBMC. For MM6 cells, the concentration-dependence of taurolidineinhibition was biphasic, with high concentrations strongly inhibitingTNFα expression and intermediate concentrations (most particularly, 25μg/ml) markedly stimulating TNFα secretion. Taurine at 44 μg/ml had noeffect on TNFα expression by MM6 cells.

Treating peripheral blood monocytes or cultured MM6 cells withtaurolidine strongly inhibited the induction of TF in response to LPS.Expression of TF is an important effector function of activatedmonocytes in sepsis and, in particular, previous studies have shown thatblocking antibodies to TF can protect baboons from the lethal effects ofintravenous E. coli in an animal model of gram-negative septic shock(Taylor, et al., “Lethal E. coli septic shock is prevented by blockingtissue factor with monoclonal antibody,” Circ Shock 33:127 (1991)) andcan attenuate the coagulopathy associated with LPS injection intochimpanzees (Levi, et al, “Inhibition of endotoxin-induced activation ofcoagulation and fibrinolysis by pentoxifylline or by a monoclonalanti-tissue factor antibody in chimpanzees,” J Clin Invest 93:114(1994)). Therefore, the ability of taurolidine to block the induction ofTF in monocytes is an important aspect of its clinical efficacy intreating sepsis.

In order to determine if taurolidine would inhibit LPS-induced TFexpression in PBMC in their native milieu, i.e., whole blood, a tissuefactor activity assay was performed. LPS (10 ng/ml) and taurolidine(100-500 μg/ml) or equal dilutions of the taurolidine vehicle were addedto 5 ml aliquots of heparinized blood, and the blood was incubated in 50ml centrifuige tubes at 37° C., rotating on a horizontal rotator at 120rpm. After 4 hours, PBMC were purified from the blood using Histopaqueas described previously. Cells were lysed by 3 freeze-thaw cycles, andused as a source of TF in coagulation assays. For these assays, 50 μl oflysate (at 2×10⁷ cells/ml) was added to 50 μl pooled normal humanplasma, incubated for 30 seconds at 37° C., then 50 μl of 25 MM CaCl₂was added and clot formation assayed using a Diagnostica Stago ST4coagulometer. Protein content of the cell samples was determined bybicinchoninic acid assay (Pierce Chemical Company, Rockford, Ill.), andused to normalize the clotting data. A standard curve was made withdescyto TF reconstituted in PS/PC vesicles according to the method ofMimms et al. (Mimms, et al., “Phospholipid vesicle formation andtransmembrane protein incorporation using octyl glucoside,” Biochemistry20:833-840 (1981)). One unit of TF was defined as that which caused clotformation at 50 seconds of incubation with CaCl₂. The TF activity assayswere performed on LPS-induced PBMC after 4 hour treatment in wholeblood. A dose-response effect of taurolidine on monocyte TF isillustrated in Table II. Taurolidine at 100 μg/ml decreased TF activityby 12%; 200 μg/ml decreased activity by approximately 60%, and treatmentwith 500 μg/ml taurolidine resulted in loss of approximately 75% of TFactivity. Thus, higher concentrations of taurolidine were required toinhibit TF when the PBMC were treated with whole blood when compared toPBMC in culture medium. Further, lower concentrations of taurolidinewere required to down-regulate TF expression than were needed to induceapoptosis as described below.

In addition to its role in the pathology of sepsis, TF expression on thesurface of leukemia cells (especially acute myelogenous leukemias (AML)and

TABLE II Effect of Taurolidine Treatment on Coagulation Activity ofLPS-induced PBMC Treatment % of control^(a) control 100 vehicle 123.5 ±29.0 100 μg/ml 87.7 ± 6.6 taurolidine 200 μg/ml 41.7 ± 8.0 taurolidine500 μg/ml  26.5 ± 10.6 taurolidine ^(a)Heparinized blood was incubatedin the presence of LPS and varying amounts of taurolidine, then PBMCwere purified, and coagulation assays performed on cell lysates. Unitsof TF per mg protein were calculated as described above, and dataexpressed as percentage of LPS-only (“control”) samples. Data shown aremean ± S.D. of two experiments.

acute lymphoblastic leukemias (ALL) is associated with severecoagulopathies in leukemic patients (Bauer, et al., “Tissue factor geneexpression in acute myeloblastic leukemia,” Thromb Res 56:425-430(1989); Hair, et al., “Tissue factor expression in human leukemiacells,” Leuk Res 20:1-11 (1996); and Tanaka, M. and Yamanishi, H., “Theexpression of tissue factor antigen and activity on the surface ofleukemic cells,” Leuk Res 17:103-111 (1993). These coagulopathies caninclude life-threatening thrombotic and bleeding episodes, as well asthe development of disseminated intravascular coagulation (DIC).Furthermore, episodes of thrombosis and DIC in such leukemic patientscan be induced by chemotherapy. Therefore, the ability of taurolidine toreduce expression of TF by leukemic cells (exemplified by the MM6 cellline) should be beneficial in reducing the incidence and severity ofcoagulopathies in leukemic patients, even at doses which do not kill theleukemia cells.

Effect of Taurolidine on Cell Viability and Growth Rates

Apoptosis is a controlled form of cell death characterized by the factthat neither parent cells nor apoptotic bodies becomemembrane-permeable. This characteristic distinguishes apoptosis fromnecrosis wherein cell death involves rupture of cell membranes.Assessment of apoptosis was performed by determining the cell membraneintegrity of various cell lines, continued long-term culture of cellsafter taurolidine treatment to determine growth rate, and membranepermeability after taurolidine treatment. It was found that taurolidinecauses apoptosis rather than necrosis of leukemia cells.

In these experiments, integrity of cell membranes following taurolidinetreatment was assessed by exclusion of trypan blue and by measuring therelease of LDH in MM6, K-562, HL-60, and REH cells. Since BCS containssignificant amounts of LDH, these experiments were conducted on cellscultured in AIM-V+0.01% BCS. Four hour treatment of cells was conductedin AIM-V containing 0.01% BCS in order to allow subsequent LDH assaywithout interference from BCS (which occurs in when normal culturemedium, containing 10% BCS, is used). Thus, MM6 cells (5×10⁵ cells/ml)were treated with taurolidine for four hours in AIM-V+0.01% BCS, afterwhich an aliquot of the cell suspension was removed for measurement ofLDH release. A parallel aliquot of cells was lysed by three cycles offreeze/thaw and the quantity of LDH released was taken to be 100%.Remaining cells were rinsed in HBSS and resuspended in growth medium formeasurement of growth rates.

Upon microscopic examination of MM6 cultures treated for four hours with100 μg/ml taurolidine, the cells appeared to be smaller than controlcells and the cultures were observed to contain numerous small,spherical bodies which excluded trypan blue. In one study, the integrityof the cell membrane was assessed by measurement of LDH release in PBMCand MM6 cells cultured in AIM-V+0.01% BCS. After treatment with 0 to 50μg/ml taurolidine for four hours, LDH levels in the treated culturesranged from 6 to 9% of that of freeze/thaw lysed cells (mean values ofthree separate experiments), which was not significantly different fromLDH levels in cultures not treated with taurolidine. Additionally, afterfour hour treatment, control and taurolidine-treated cells were equallyable to exclude trypan blue (FIG. 4A, Day 1). However, whentaurolidine-treated MM6 cells were rinsed and resuspended in growthmedium, 65% of cells exposed to 50 μg/ml taurolidine became trypanblue-positive on the day following treatment (FIG. 4A, Day 2),indicating that the plasma membrane had become permeable. In contrast,only 5% of untreated (“control”) and vehicle-treated cells were trypanblue-positive. Cells treated with 50 μg/ml taurolidine for four hoursfailed to grow when returned to growth medium, while cells treated withvehicle alone or with 25 μg/ml taurolidine exhibited growth ratessimilar to that observed with untreated cells (FIG. 4B). In anotherstudy, K-562, HL-60, and REH cells were tested after a four hourtreatment with taurolidine, and the results are summarized in Table III.

HL-60 cells treated with 50 μl taurolidine. In these samples, LDHpresent in the culture medium was 8.3±2.3% of that released bycompletely lysed cells, compared with 2.3±0.8% measured in controlcultures. All samples from all other cell lines displayed LDH levelswhich were ≦7% of lysed cell samples (n=3). After 4 hour treatment,cells were rinsed and returned to growth medium and monitored forviability and growth over the following 3 days. Concentrations oftaurolidine from 25 μg/ml to 100 μ/ml were used in order to highlightdifferences in sensitivity of cell lines to taurolidine. Trypan blueassay of all samples were equivalent to control on Day 1 (after 4 hoursof treatment) indicating that taurolidine treatment did not cause rapidpermeability of cell membranes, suggesting that this compound did notcause cellular necrosis. However, over the following days, allleukemia-derived-cell lines demonstrated progressive

TABLE III Results from a Four Hour Treatment with Taurolidine Conc. LDHgrowth rate Cell Type Taurolidine (% of total)^(a) (% of control)^(b)K-562 (chronic control 8 100 myeologenous  50 μg/ml 6  52 leukemia) 100μg/ml 8  0 vehicle 6  80 HL-60 (acute control 5 100 myeloid  50 μg/ml 5 0 leukemia) 100 μg/ml 6  3 vehicle 4  92 REH (acute control 4 100lymphocytic  50 μg/ml 6  1 leukemia) 100 μg/ml 5  18 vehicle 5 119^(a)LDH was measured immediately after four hour taurolidine treatment.^(b)Growth rate was measured over three days following treatment.

permeability to trypan blue after treatment with concentrations oftaurolidine of 50 μg/ml or greater, with the exception of K562 cells(FIGS. 5A, 6A, 7A, 8A, and 9A). MM6, K562, and Jurkat cells did not showan increase in trypan blue staining after treatment with 25 μg/mltaurolidine, while HL-60 and REH cells were more sensitive. Asexplained, eventual permeability of cell membranes is inevitable in invitro apoptosis. K562 cells demonstrated an inhibition of growth aftertaurolidine treatment of 50 μg/ml or greater as did the otherleukemia-derived cell lines (FIGS. 5B, 6B, 7B, 8B, and 9B). Cell linesdemonstrated differing sensitivities to taurolidine in regard to cellgrowth, with K562 and Jurkat cells being least affected, and HL-60growth being most inhibited after taurolidine treatment.

The adherent cell lines ECV304 (endothelial cells) and GM5387(fibroblasts) did not demonstrate a significant increase in trypan bluestaining of cells during 4 day growth after taurolidine treatment (FIGS.10A and 11A). However, proliferation of these cells was inhibited aftertreatment with 50 and 100 μg/ml taurolidine (FIGS. 10B and 11B).

Detection of Apoptosis

The effect of taurolidine on cell lines of various lineages with respectto apoptosis was evaluated. Treatment with effective amounts oftaurolidine resulted in apoptosis in a variety of monocytic,granulocytic and numerous leukemic cell lines, but not in lymphocytes.

Following treatment with either taurolidine or the vehicle solution,cell death was assessed in one of two ways. In the first method, the DNAfragmentation pattern characteristic of apoptosis was detected byextracting low molecular weight DNA from cells using a phosphate-citratebuffer (Darzynkiewicz, et al., “Assays of cell viability: discriminationof cells dying by apoptosis,” Methods Cell Biol 41:15 (1994)) andsubjecting the extracted DNA to agarose gel electrophoresis.

In the second method, DNA fragmentation was detected via TUNEL assayaccording to the manufacturer's instructions. With this methodology,2×10⁶ cells were fixed with 2% paraformaldehyde, permeabilized with 0.1%Triton X-100/0.1% citrate, incubated at 37° C. with terminaldeoxynucleotidyl transferase and FITC-dUTP, rinsed and analyzed by flowcytometry. Enhanced FITC signal indicates apoptosis-specific DNAfragmentation. Gavriali, et al, “Identification of programmed cell deathin situ via specific labeling of nuclear DNA fragmentation,” J Cell Biol119:493 (1992). Parallel aliquots of unfixed cells were treated with PIand FITC-conjugated anti-CD14 and analyzed by flow cytometry, in orderto identify cells with permeable cell membranes (since PI binds to DNAbut is excluded from cells with intact plasma membranes) and also todetermine the cell-specificity of the apoptotic signal.

A Becton-Dickinson FACSCalibur instrument was used for flow cytometricanalysis of cells. Assessment of cell diameter (forward scatter; FCS)and granularity (side scatter; SSC).was carried out on unfixed cells.Discrimination between lymphocytes and monocytes was made possible bytheir characteristic differences in FSC and SSC, and confirmed bystaining monocytes with FITC-labeled anti-CD14 antibodies andlymphocytes with a combination of FITC-conjugated anti-CD3 andPE-conjugated anti-CD19. After incubation with taurolidine (with orwithout concomitant LPS treatment), cells were rinsed twice inphosphate-buffered saline containing 0.1% bovine serum albumin (PBSA)and 1×10⁶ cells were resuspended in 0.1 ml PBSA. After 5 μlFITC-conjugated anti-CD14 was added, cells were incubated in the dark onice for 15 minutes and then centrifuged at 250×g. Cells were resuspendedin 500 μl PBSA containing 5 μg/ml PI and subjected to flow cytometry.

Taurolidine-treated MM6 cells and peripheral blood monocytes displayedreduced cell size as illustrated by a decrease in FSC and SSC when cellswere analyzed by flow cytometry (FIG. 12). After a three hour treatmentwith taurolidine, MM6 cells demonstrated an approximately 30% decreasein FSC, as did peripheral blood monocytes after a six hour treatment.These morphological changes (reduced cell size and appearance ofspherical bodies) were concomitant with an intact plasma membrane,indicating that taurolidine induces apoptosis in monocytic cells.

Apoptosis is associated with fragmentation of genomic DNA in theinternucleosomal linker regions, which can be visualized as acharacteristic ladder of DNA fragments upon gel electrophoresis. Arends,M. J. and Wyllie, A. H., “Apoptosis: mechanisms and roles in pathology,”Int Rev Exp Path 32:223 (1991). Accordingly, low molecular weight DNAwas extracted from taurolidine-treated MM6 cells and subjected toelectrophoresis in agarose gels. Cells treated with 50 lμg/mltaurolidine for four hours exhibited a prominent ladder pattern of DNAfragments, while untreated and vehicle-treated cells did not (FIG. 13).These cells had not been treated with LPS; however, identical resultswere seen when LPS was present during taurolidine treatment. Theseresults support the conclusion that taurolidine induces apoptosis in MM6cells.

In order to examine if taurolidine treatment induced apoptosis inperipheral blood monocytes, a technique was employed for evaluating DNAfragmentation that could be applied to mixed populations of monocytesand lymphocytes. Consequently, TUNEL analysis was performed on PBMC at3, 6, or 24 hours after treatment with 25 to 100 μg/ml taurolidine.Aliquots of harvested cells were also labeled with FITC-conjugatedanti-CD14 antibodies and PI, which allowed identification of monocytes(CD14-positive cells) and cells with damaged cell membranes (PI-positivecells). While relatively few of the PBMC were TUNEL-positive after athree hour incubation, a substantial population of TUNEL-positive cellswas readily apparent after six hours of incubation with 100 μg/mltaurolidine (events to the right of the vertical line in FIG. 14),concomitant with a large loss of TUNEL-negative monocytes (region R1 inFIG. 14; see also Table IV). Thus, nearly 75% of the monocytes wereTUNEL-positive following treatment with 100 μg/ml taurolidine for sixhours. Confirmation that the TUNEL-positive cells were monocytes wasobtained by staining with FITC-conjugated anti-CD14. Lower doses oftaurolidine (25 and 50 μg/ml) resulted in lower levels of induction ofapoptosis (Table IV). Taurolidine-treated and control cells exhibitedequivalently low levels of PI-positive cells at six hours, indicatingthat the cell membranes remained intact at this time (Table IV). Inanother study summarized in Table V, taurolidine-treatedpolymorphonuclear cells (granulocytes) at 100 μg/ml, MM6 cells(monocytic leukemia cell line) at 50 μg/ml, and acute myeloid leukemiacell line (HL-60) at 50 μg/ml exhibited a high level of apoptosis asindicated by the low percentage of % TUNEL-negative cells, and the lowpercentage of propidium iodide positive cells confirmed that the treatedcells remained intact.

In contrast to its effect on monocytes, treatment with up to 100 μg/mltaurolidine for six hours did not induce detectable apoptosis in thelymphocyte

TABLE IV Dose-Response of PBMC to Taurolidine: 6 Hour Treatment^(a) %Propidium % TUNEL- Cell Type Treatment Iodide Positive Negative^(b) AllCells Control 11.0 ± 1.6  25 μg/ml taurolidine  9.0 ± 3.2  50 μg/mltaurolidine 12.2 ± 5.6 100 μg/ml taurolidine  9.0 ± 3.5 MonocytesControl 100  25 μg/ml taurolidine 88.3 ± 2.7   50 μg/ml taurolidine 67.8± 18.0 100 μg/ml taurolidine 25.1 ± 11.8 Lymphocytes Control 100  25μg/ml taurolidine 100.2 ± 7.0   50 μg/ml taurolidine 99.8 ± 3.4  100μg/ml taurolidine 101.9 ± 3.1  ^(a)Analysis by flow cytometry afterstaining cells with FITC-conjugated anti-CD14 and PI, and processing forTUNEL. Data are mean ± SD of two experiments. ^(b)Percent of cellspresent in the TUNEL-negative region in control samples.

population of PBMC, with essentially 100% of the cells remainingTUNEL-negative (FIG. 14 and Table IV).

Taurolidine at 100 μg/ml induced apoptosis of approximately 75% ofpurified peripheral blood monocytes when these cells were incubated inculture medium. However, in order to more closely mimic the milieu ofleukocytes in vivo, induction of apoptosis in PBMC by taurolidine wasstudied in whole blood (anticoagulated with heparin). As before,apoptosis was quantitated by measuring the loss of monocytes from aTUTNEL-negative (no DNA fragmentation), CD 14 positive population ofcells which was concomitant with the appearance of a TUNEL-positivepopulation of cells. Treatment of PBMC in whole blood resulted

TABLE V Induction of Apoptosis by Taurolidine % Propidium % TUNEL- Conc.Length of Iodide Positive Negative Cell Type Taurolidine IncubationCells^(a) Cells^(b) granulocytes^(c) control 6 hours 1 98 100 μg/ml 6hours 2  8 Mono Mac 6^(d) control 3 hours 10  98  50 μg/ml 3 hours 7 44HL-60^(e) control 3 hours 7 85  50 μg/ml 3 hours 5 35 ^(a)Propidiumiodide labels only those cells with leaky plasma membranes. Apoptoticcells do not initially have leaky plasma membranes. ^(b)The TUNEL assayis used to identify cells with apoptosis-specific DNA fragmentation.TUNEL-negativecells are not apoptotic. ^(c)granulocytes(polymorphonuclear cells). ^(d)Monocytic leukemia cell line. ^(e)Acutemyeloid leukemia cell line.

in a shift of the taurolidine dose-response curve. Concentrations oftaurolidine up to 200 μg/ml did not induce significant apoptosis whencells were treated in whole blood (Table VI and FIG. 15A). However,taurolidine at 500 μg/ml resulted in the appearance of a population ofTUNEL-positive cells (FIG. 15B, region 3), and loss of 70% ofTUNEL-negative monocytes (Table VI). As a positive control for inductionof apoptosis, some cells were treated with anisomycin, a proteinsynthesis inhibitor known to induce apoptosis in monocytes. Anisomycintreatment of PBMC in whole blood also resulted in a loss ofTUNEL-negative monocytes; however, fewer TUNEL-positive cells weredetected after 6 hour incubation (FIG. 15C, region 3). There was noevidence for a significant effect of either taurolidine or anisomycin oninduction of apoptosis in lymphocytes.

TABLE VI Dose-response of PBMC (in whole blood) to Taurolidine^(a)Treatment % propidium iodide % TUNEL-negative^(b) All cells control 1.4± 0.6 200 μg/ml taurolidine 1.6 ± 0.6 500 μg/ml taurolidine 1.4 ± 0.2Monocytes control 100 200 μg/ml taurolidine 117.4 ± 11.2 500 μg/mltaurolidine  27.9 ± 12.0 Lymphocytes control 100 200 μg/ml taurolidine92.7 ± 6.0 500 μg/ml taurolidine 91.8 ± 6.9 ^(a)Analysis by flowcytometry after staining cells with PI and processing for TUNEL. Dataare mean ± SD of 2 experiments. ^(b)Percent of cells present in theTUNEL-negative region in control samples.

The effect of taurolidine on granulocytes was assayed by TUNEL analysison taurolidine-treated purified granulocytes. These experiments wereconducted in culture medium rather than whole blood. Granulocytes weretreated with 100 μg/ml taurolidine since it had been shown that thisconcentration of taurolidine is apoptogenic to peripheral bloodmonocytes treated in culture medium. TUNEL-positive granulocytes rangedfrom 2% to 30% in untreated samples, and varied according to blooddonor. Taurolidine treatment resulted in apoptosis of 92% of thegranulocytes within 6 hours (Table VII). Therefore, the apoptogeniceffect of taurolidine is not specific to monocytic cells.

TABLE VII Response of Granulocytes to 100 μg/ml Taurolidine^(a)granulocytes % propidium iodide positive % TUNEL-positive control 0.5 ±0.2 17.0 ± 7.0 taurolidine 1.6 ± 0.5 91.5 ± 3.5 vehicle 0.5 ± 0.2 17.5 ±7.0 ^(a)Analysis by flow cytometry after staining cells with PI andprocessing for TUNEL. Data are mean ± SEM of 5 experiments.

In another study, TUNEL assays were performed on all cell lines 6 hoursafter cells were initially exposed to taurolidine (cells were treatedfor 4 hours, rinsed, and then incubated for 2 more hours before TUNELassay was performed).

For these experiments, the effect of 100 μg/ml taurolidine was comparedto that of 1 μg/ml anisomycin, a protein synthesis inhibitordemonstrated to induce apoptosis in monocytic (Kochi, S. K. and Collier,R. J., “DNA fragmentation and cytolysis in U937 cells treated withdiphtheria toxin or other inhibitors of protein synthesis,” Exp Cell Res208:296-302 (1993)), granulocytic (Polverion, A. J. and Patterson, S.D., “Selective activation of caspases during apoptotic induction inHL-60 cells,” J Biol Chem 272:7013-7021 (1997)), and epithelial (Liao,et al., “Stress, apoptosis, and mitosis induce phosphorylation of humankeratin 8 at Ser-73 in tissues and cultured cells,” J Biol Chem272:17565-17573 (1997)) cell lines. Taurolidine and anisomycin inducedapoptosis in MM6, HL-60, and Jurkat cells (Table VIII). MM6 and HL-60cells were similarly effected, with >80% TUNEL-positive cells after 6hours. Jurkat cells were less sensitive, with approximately 50% of cellsinduced to undergo apoptosis after taurolidine treatment. After 6 hours,73% of anisomycin-treated REH cells were apoptotic, with less than 20%of taurolidine-treated REH cells being TUNEL-positive. However, assay ofcells 24 hours after

TABLE VIII Response of Cell Lines to Taurolidine and Anisomycin^(a) Cellline % propidium iodide positive % TUNEL-positive MM6 control 7.3 ± 0.53.6 ± 2.6 taurolidine 22.4 ± 0.8  80.9 ± 7.7  anisomycin 19.1 ± 5.4 92.0 ± 2.3  K562 control 2.2 ± 0.1 0.6 ± 0.2 taurolidine 2.2 ± 0.8 6.6 ±2.8 anisomycin 4.8 ± 3.1 8.5 ± 8.8 HL-60 control 3.7 ± 3.2 2.4 ± 0.6taurolidine 9.1 ± 8.2 91.9 ± 10.9 anisomycin 28.3 ± 15.3 96.1 ± 6.8  REHcontrol 2.5 ± 0.6 2.8 ± 1.0 taurolidine 4.4 ± 1.6 18.3 ± 10.7 anisomycin18.4 ± 0.5  72.9 ± 7.4  Jurkat control 3.6 ± 0.3 0.6 ± 0.2 taurolidine6.1 ± 5.8 51.7 ± 1.4  anisomycin 5.1 ± 3.4 69.6 ± 19.8 ECV304 control5.6 ± 0.7 0.0 ± 0.0 taurolidine 2.0 ± 1.4 0.0 ± 0.0 anisomycin 2.2 ± 3.10.0 ± 0.0 GM5387 control 5.7 ± 2.1 0.0 ± 0.0 taurolidine 5.9 ± 1.8 0.0 ±0.0 anisomycin 5.4 ± 3.2 0.0 ± 0.0 ^(a)Analysis by flow cytometry afterstaining cells with propidium iodide and processing for TUNEL. Data arepercent of total cell-size events, and are expressed as mean ± SD of twoexperiments. Cells were treated for 4 hours with taurolidine (100 μg/ml)or anisomycin (1 μg/ml), and then assayed at 6 hours.

treatment indicated that taurolidine does induce apoptosis in REH cells(Table IX), but that it occurs less rapidly than with anisomycintreatment, suggesting that these two agents may work by differentmechanisms. K562 cells were the most resistant to apoptosis, with lessthan 10% TUNEL-positive cells 6 hours after treatment with eitheranisomycin or taurolidine. Some induction of apoptosis was demonstratedafter 24 hours, with approximately 20% of taurolidine-treated K562becoming TUNEL-positive.

Less than 0.1% of ECV304 or GM5387 cells were TUNEL-positive 6 hoursafter treatment with taurolidine or anisomycin (Table VIII). Therefore,neither of these agents induce rapid DNA fragmentation in these celltypes. However, there was approximately a 70% increase in the number ofsubcellular-sized events recorded by flow cytometry after taurolidinetreatment of these cells, indicating some effect of taurolidine on cellintegrity. These cell fragments were not stained by propidium iodide,consistent with them being apoptotic bodies.

In summary, concentrations of taurolidine that were effective ininhibiting the induction of TF and TNFα expression in LPS-stimulatedmonocytic cells were also effective in inducing apoptosis in thesecells. This was established by agarose gel electrophoretic analysis ofDNA fragmentation MM6 cells and by TUNEL

TABLE IX Response of Cell Lines to Taurolidine and Anisomycin: Apoptosisat 24 hours After Treatment^(a) Cell line % propidium iodide %TUNEL-positive K562 control 2.2 ± 0.3 0.5 ± 0.2 taurolidine 12.4 ± 2.1 21.2 ± 14.6 anisomycin 7.1 ± 2.3 2.2 ± 6.2 REH control 3.6 ± 0.2 1.7 ±1.8 taurolidine 55.8 ± 19.9 81.4 ± 4.4  anisomycin 51.6 ± 17.0 53.0 ±2.6  ^(a)Analysis by flow cytometry after staining cells with propidiumiodide and processing for TUNEL. Data are percent of total cell-sizedevents, and are expressed as mean ± SD of two experiments. Cells weretreated for 4 hours with taurolidine (100 μg/ml) or anisomycin (1μg/ml), and then assayed at 24 hours.

analysis of peripheral blood monocytes. Induction of apoptosis wasrelatively rapid, being detectable within four to six hours aftertreatment in MM6 cells and monocytes. In this time frame, DNAfragmentation was readily detectable in cells whose plasma membraneswere still intact. Eventually, however, cell permeability increased(e.g., 24 hours after treatment with taurolidine). This is likely to bedue to “secondary necrosis,” an inevitable result of advanced in vitroapoptosis where there is no system for phagocytosis and intracellulardegradation of apoptotic cells. It has now been found that taurolidinealso induces apoptosis of monocytic cells treated in whole blood. Theeffective concentration (500 μg/ml) is 5 times that required whenpurified PBMC are treated in culture medium. It has been proposed thatmetabolites of taurolidine inactivate LPS and kills bacteria by formingcross-links between primary amine and hydroxyl groups on LPS andbacterial cell walls. (Browne, et al., “Taurolin, a new chemotherapeuticagent,” J Appl Bacteriol 41:363-368 (1976); Pfimann, R. W., “Taurolin:ein neues konzept zur antimikrobiellen chemotherapie chirurgischerinfektionen ein fuhrung und ubersicht,” In: Taurolin, ein neues konzeptzur antimikrobiellen chemotherapie chirurgischer infektionen, edited byBrückner, W. L. and Pfirrmann, R. W., München-Wien-Baltimore: Urban andSchwarzenburg; pp. 3-23 (1985)). If taurolidine induces apoptosis bysimilar mechanisms, incubation in whole blood could reduce itseffectiveness by providing a high concentration of non-cellularsubstrates. In fact, taurolidine has been reported to react withproteins in plasma and serum (Jones, et al., “A study of the stabilityof taurolidine in plasma and protein-free serum,” Int J Pharm 64:R1-R4(1990)). Taurolidine also reduces the TF activity of LPS-inducedmonocytes treated in whole blood, with significant reduction seen withconcentrations of taurolidine as low as 200 μg/ml.

Taurolidine does not induce a significant amount of apoptosis inlymphocytes even at concentrations (in whole blood) up to 500 μg/ml.However, taurolidine is at least as effective in inducing apoptosis ingranulocytes as it is in monocytes, as measured by TUNEL assaysperformed on purified cells treated in culture medium. It has beenreported that both granulocytes and monocytes, but only a fraction oflymphocytes, constitutively express Fas antigen (Iwai, et al.,“Differential expression of bcl-2 and susceptibility toanti-Fas-mediated cell death in peripheral blood lymphocytes, monocytes,and neutrophils,” Blood 84:1201-1208 (1994)), and that cross-linking ofthe cell surface Fas mediates transduction of apoptotic signals intocells (Itoh, et al., “The polypeptide encoded by the cDNA for human cellsurface antigen Fas can mediate apoptosis,” Cell 66:233-243 (1991)).Although the mechanism by which taurolidine induces apoptosis isunknown, taurolidine affects monocytes and granulocytes but notlymphocytes.

Specificity of taurolidine's ability to induce apoptosis was furtherinvestigated by assessing its effects on a variety of leukemic celllines. Taurolidine was most effective in inducing apoptosis in monocytic(MM6) and granulocytic (HL-60) leukemic cell lines, analogous to itspotent effect on peripheral blood monocytes and granulocytes. However,taurolidine was also able to induce apoptosis in a significantproportion of Jurkat cells, a lymphocytic leukemia cell line.Approximately 50% of these cells became apoptotic within 6 hours oftreatment. Taurolidine did not induce apoptosis in peripheral bloodlymphocytes under similar experimental conditions, indicating thatleukemia-derived cells may be more sensitive to taurolidine than normalcells. Taurolidine was also able to induce apoptosis in thelymphoblastic cell line REH, although the process in these cells wasdelayed as compared to MM6 and HL60.

As noted in previous studies (Evans, et al., “Activation of the Abelsontyrosine kinase activity is associated with suppression of apoptosis inhemopoietic cells,” Cancer Res 53:1735-1738 (1993)), the multipotentialcell line K562 was relatively resistant to apoptosis. Anisomycin inducedless than 10% of these cells to become TUNEL-positive. Taurolidine wassomewhat more potent, with approximately 20% of the cells becomingTUNEL-positive 24 hours after treatment was begun.

Although taurolidine inhibited proliferation of the endothelial andfibroblast cell lines, strong evidence for induction of apoptosis inthese cells was not obtained. Taurolidine treatment did not causemeasurable cell death, as shown by the fact that cells remained trypanblue negative throughout the 4 day culture following treatmentAdditionally, these cells did not demonstrate evidence for fragmentationof DNA (TUNEL positivity) when assayed 6 hours after treatment. DNAfragmentation has been used to assay for apoptosis in fibroblast celllines (Kim, et al., “Platelet-derived growth factor induces apoptosis ingrowth-arrested murine fibroblasts,” Proc Natl Acad Sci USA 92:9500-9504(1995))., including, in particular, the TUNEL assay (Gansauge, et al.,“The induction of apoptosis in proliferating human fibroblasts by oxygenradicals is associated with a p53- and p21^(WAF1CIP1) induction,” FEBSLetters 404:6-10 (1997)). However, some fibroblast cell lines have notexhibited DNA degradation, and apoptosis was identified by changes incell morphology and detachment of cells from the culture vessel (Boyle,et al., “Apoptosis in C3H-10T1/2 cells: roles of intracellular pH,protein kinase C, and the Na+/H+ antiporter,” J Cell Biochem 67:231-240(1997)). Additionally, these studies examined cells for evidence ofapoptosis 24-48 hours after treatment with the apoptogenic agent. Whileevidence for DNA fragmentation of fibroblast or endothelial cells wasnot found, analysis by flow cytometry did reveal an increase in cellfragmentation.

Taurolidine induces apoptosis in monocytes, granulocytes, and numerousleukemic cell lines. In all cells tested, it was as efficient anapoptogenic agent as the protein synthesis inhibitor anisomycin.Intravenous administration of taurolidine does not cause detectablealterations in hematologic variables, including white cell count(Browne, M. K., “Pharmacological and clinical studies with taurolin.” InTaurolin, Ein Neues Konzept zur Antimikrobiellen ChemotherapieChirurgischer Infektionen, Brückner, W. L. and Pfirrmann, R. W. (eds),München-Wien-Baltimore, Urban & Schwarzenberg, p. 51-60 (1985); Browne,et al., “A controlled trial of taurolin in established bacterialperitonitis,” Surg Gynecol Obstet 146:721-724 (1978); and Williats, etal., “Effect of the antiendotoxic agent, taurolidine, in the treatmentof sepsis syndrome,” Crit Care Med 23:1033-1039 (1995)). Taurolidineprovides a treatment for a variety of leukemic diseases due to itsability to induce apoptosis in leukemic cells and cells in whole blood,in conjunction with the absence of a generalized toxicity in humansubjects.

Induction of apoptosis in monocytic cells by taurolidine appeared to beindependent of any effects the drug may have on LPS. In fact,taurolidine induced apoptosis in monocytic cells in the absence of LPStreatment. Thus, in addition to its potential utility as a treatment forsepsis previously reported by others, we have found that taurolidine isa very effective, selective inducer of apoptosis in monocytic andmyeloid cells and has utility in the treatment of certain leukemias,including monocytic and mycloid leukemias.

EXAMPLE 1 In Vitro Screening Test for Taurolidine Treatment

An in vitro screening test is performed on a patient's blood todetermine if the monocytic, myeloid and/or leukemia cells would respondto taurolidine therapy by exhibiting apoptosis.

A patient's peripheral blood leukocytes are isolated from heparinizedblood by centrifugation using Histopaque, Ficoll-Hypaque, or any othersuitable methodology. After separation, the isolated leukocytes aresuspended in RPMI+10% BCS. Aliquots ofthe suspended cells are treatedwith 10-100 μg/ml taurolidine for 4-6 hours. Cells treated with thevehicle solution alone are used as controls. The presence of apoptosisis measured as the degree of DNA fragmentation using either the agarosegel electrophoresis or the TUNEL assay. A high degree of apoptosisobserved in vitro with the patient's leukemia cells is indicative of afavorable prognosis upon in vivo taurolidine treatment, while a minimaldegree of apoptosis suggests alternative treatment might be preferable.

EXAMPLE 2 In Vivo Taurolidine Treatment

To decrease the number of monocytic andlor myeloid leukemia cells, apatient with monocytic and/or myeloid leukemia is treated with about 10to 500 mg/kg body weight taurolidine administered either intravenouslyor intraperitoneally. The patient's response to treatment is monitoredby a decrease in WBC count and examining the patient's leukocytes for adecrease in leukemia cells. Treatment can be continued as necessary.

EXAMPLE 3 In vivo Taurolidine Treatment to Reduce Coagulopathies

To decrease the hypercoagulable state associated with TF expression oncirculating leukemia cells, a patient with leukemia and an associatedcoagulopathy is treated with about 10 to 500 mg/kg body weighttaurolidine administered either intravenously or intraperitoneally. Inaddition, treatment can be given prior to, or concurrently with,standard cancer chemotherapy, in order to reduce the incidence orseverity of thrombotic complications. The patient's response totreatment is monitored by any of a variety of standard hematologicmeasures of hypercoagulability or DIC. These measures includeprolongation of the prothrombin time, elevated levels of fibrindegradation products (FDP), elevated levels of fibrin D-Dimer, andhypofibrinogenemia. A positive response to treatment is indicated by areturn of these measures toward normal (control) levels. Treatment canbe continued as necessary.

What is claimed is:
 1. A method for treating a leukemia patient withtaurolidine consisting essentially of the steps of administering to saidpatient an effective amount of taurolidine to render leukemia-causingcells in said patients apoptotic.
 2. The method of claim 1, wherein saidamount of taurolidine is administered intravenously.
 3. The method ofclaim 1, wherein said amount of taurolidine is admniisteredintraperitoneally.
 4. The method of claim 1, wherein said effectiveamount of taurolidine is from about 10 to about 500 milligrams perkilogram body weight.
 5. The method of claim 1, wherein said effectiveamount of taurolidine is about 150 milligrams per kilogram body weight.6. A method for treating a leukemia patient with taurolidine, saidpatient exhibiting coagulopathy caused by TF expression on leukemiacells, comprising the steps of administering to said patient aneffective amount of taurolidine to reduce coagulopathy in said patient.7. A method of treating a leukemia patient with taurolidine, saidpatient exhibiting coagulopathy caused by TF expression on leukemiacells, consisting essentially of the steps of administering to saidpatient an effective amount of taurolidine to reduce coagulopathy insaid patient.
 8. The method of claim 6, wherein said amount oftaurolidine is administered intraperitoneally.
 9. The method of claim 6,wherein said effective amount of taurolidine is from about 10 to about500 milligrams per kilogram body weight.
 10. The method of claim 6,wherein said effective amount of taurolidine is about 150 milligrams perkilogram body weight.
 11. The method of claim 7, wherein said effectiveamount of taurolidine is about 150 milligrams per kilogram body weight.12. A method of rendering leukemia cells apoptotic consistingessentially of contacting said cells with an effective amount oftaurolidine.
 13. A method of decreasing the hypercoagulable stateassociated with TF expression on leukemia cells consisting essentiallyof contacting said cells with an effective amount of taurolidine.